Alpha-fetoprotein (AFP) is a major transport protein in the fetus, acting as a shuttle and having a halflife of 3-5 days (Mizejewski, G. J., in “AFP and Congenital Disorders”, pp. 5-34, Academic Press, Orlando, 1985; Abelev, G I, Alpha-fetoprotein: 25 years of study, Tumor Biology, 10:63-74; 1989). Expression of AFP is tightly regulated during development such that detectable levels of AFP expression are largely dependent on the developmental stage. Studies have established that AFP acts as a growth regulator during both ontogenic growth and tumour progression. Due to expression of AFP during development and tumourigenesis, AFP is referred to as an oncofetal antigen.
AFP is a glycoprotein belonging to the albuminoid gene superfamily, of which albumin is also a member. The molecular weight of AFP can vary from 64,000 to 72,000 daltons depending on the source, developmental stage and the method used for its purification. Associated percentage of carbohydrate varies from 3% to 5% again depending on the source and developmental stage.
AFP appears to be present in two basic molecular forms: 1) an unbound form, and 2) a bound form in which AFP is complexed to various ligands (e.g. fatty acids, estrogens, phytosteroids). However, variant forms of AFP have been identified. Different conformations (holoforms) of bound AFP exist which are dependent on the nature and concentration of the bound ligand(s). Molecular variants of Human AFP (HAFP) have been identified wherein the variations are attributed to carbohydrate microheterogeneity (i.e. different carbohydrate moieties bind at the site of glycoslation on HAFP) as well as due to differences in isoelectric points (Keel, B. A., et al., CRC Press; vol 2, 24-31, 1989; Mizejewski, G. J., Exp. Biol. Med. 226(5):377-408, 2001; Morinaga, T. et al., Proc. Natl. Acad. Sci. USA, 80(15):4604-8, 1983; Parker, M. H. et al., Purification and characterization of a recombinant version of human AFP expressed in the milk of transgenic goats, Protein Expression and Purification, 38:177-183, 2004). Genetic variants of HAFP have been detected that are attributed to developmental phase-specific expression of HAFP mRNA.
Mizejewski G. J. et al. (Tumour Biol. 7(1): 19-36, 1986) describe the cyclic physiology of AFP as the “developmental clock”. The authors note that the structure and function of AFP changes throughout the course of development where the protein is expressed in fluctuating levels during fetal development and expression levels decline to negligible levels post-naturally, having a normal adult serum concentration of less than 50 ng/mL (Ruoslahti and Seppala, Int. J. Cancer 8:374-378, 1971). However, AFP plasma levels can be one thousand-fold higher in individuals with various cancers (Ruoslahti and Seppala, Adv. Cancer Res. 29:275-310, 1979). In addition, a number of cancers express high levels of AFP receptors on their cell surfaces (Uriel, J. et al., in “Biological Activities of AFP”, CRC Press, 1987, Boca Raton, Fla., vol. 2, pp. 104-117; Moro, R., in “Biological Activities of AFP”, CRC Press, 1987, Boca Raton, Fla., vol. 2, pp. 120-127). Therefore, in humans, AFP functions as a tumor marker in addition to being a fetal defect marker during embryogenesis.
Various chemical preparations, such as alkylating agents, antimetabolites, alkaloids, antibiotics, hormones and immunomodulators, known in the prior art are used to treat cancer. However, these preparations do not specifically target tumor cells resulting in what is referred to as “bystander effect”, where normal, non-tumour cells are also susceptible to the anti-cancer agent. The overexpression of HAFP receptors (HAFPR) on the surface of malignant cells, compared to negligible expression of receptors on normal cells, prompted research into the use of HAFP as a carrier/transporter of anticancer drugs (Severin, S. E. et al., Biochem. Mol. Biol. Int. 37(2):385-92, 1995; Severin, S. E. et al., Dokl. Akad. Nauk 366(4): 561-4, 1999) to target cancer cells specifically. It has been demonstrated that HAFP can target anticancer drug conjugates to tumor cells (Moskaleva et al., Cell Biol Int. 21(12):793-799, 1997; Sotnichenko et al., FEBS Letters 450:49-51, 1999; U.S. Pat. No. 6,630,445 to Murgita). The high specificity of HAFP for cancer cells that bear receptors for AFP provides enhanced efficacy of drugs due to specific targeting to tumour cells. In addition, such modes of active agent delivery are safer for the patient as normal surrounding cells are spared.
HAFP bound with numerous anticancer drugs including doxorubicin, daunomycin, calichemicin, carboxyphosphamide, bleomycetin, chlorbutin, cis-platinum, methotrexate and caminomycin has been reported (Moskaleva et al., Cell Biol. Int. 21(12):793-799, 1997; Lutsenko et al., Tumor Biology 21(6):367-374, 2000). In these instances, the active agents were bound to HAFP using chemical conjugation methods, resulting in the covalent binding of HAFP to the anticancer agent. The optimal molar ratio of AFP:drug for AFP-drug conjugates that enables both binding of ingredients without loss of their biological activity and targeted delivery of the drug was found to be 1:2 (Feldman, N. B. et al., Biochemistry 65:1140-1145, 2000). The same molar ratio 1:2 can be achieved in noncovalent binding of AFP and Dioxin (Sotnichenko et al., FEBS Letters 450:49-51, 1999).
Herve et al. (in “Biological activities of alpha-fetoprotein”, Florida Congresses, ed. Mizejewski, G. J., CRC Press, Inc., Boca Raton, Vol. 1, 1987) demonstrated warfarin and phenylbutazone binding sites on rat AFP, similar to those found on albumin. In addition, they demonstrated that these agents bind to AFP at the same large hydrophobic pocket as estrogens, fatty acids, pyrrazolic compounds and proprionic drugs. As reviewed in Mizejewski (Mizejewski, G. J., in “AFP and congenital disorders”, ed. G. J. Mizejewski, Academic Press, Inc., 1985), whereas fatty acids are capable of binding to human AFP and to rodent AFP, phytoestrogens have been shown to be capable of binding to rodent AFP only suggesting inter-species differences in AFP binding capabilities.
The predominant source of AFP used in AFP/drug targeted delivery experiments has been human AFP extracted from either female retroplacental serum (Moskaleva et al., Cell Biol Int. 21(12):793-799, 1997) or human fetal material (www.alfetin.ru). Human fetal material is difficult to obtain due to limited sources (extracted from abortion material of up to 12 weeks gestation) and it is additionally expensive. In Russia, human fetal AFP is registered as an immune modulating injectable drug under the name “Alfetinum” (1 ampoule containing 0.075 mg of 95% pure AFP). Thus, alternative sources of AFP useful in the delivery of cytotoxic agents to cancer cells would be beneficial.
Anticancer agents with different modes of action have been reported to trigger apoptosis in chemosensitive cells (Fisher, Cell 78:539-542, 1994). Changes in mitochondrial function such as mitochondrial membrane permeability and/or permeability transition pore complex alterations play a major role in apoptotic cell death including cell death induced by anticancer agents (Kroemer et al., Immunol Today 18:44-51, 1997; Susin et al., J. Exp. Med. 186:5-37, 1997; Marchetti et al., J. Exp Med. 184:1155-1160, 1996; Zamzani et al., J. Exp. Med. 183:1533-1544, 1996; Decaudin et al., Can Res 57:62-67, 1997). Many conventional chemotherapeutic agents elicit mitochondrial permeabilization in an indirect fashion by induction of endogenous effectors, such as p53, that are involved in the physiologic control of apoptosis. However, the frequent mutation of p53 in many different human cancers renders the cancer refractory to conventional chemotherapeutic agents. The discovery of cytotoxic agents that act directly on the mitochondria such as lonidamine, arsenite, betulinic acid and CD437 has provided an alternative therapeutic strategy in circumstances where conventional drugs fail due to disruption of endogenous apoptosis induction pathways, such as those involving p53 (reviewed in Costantini et al., J. Natl. Cancer Institute 92:1042-1053, 2000). Cytotoxic agents that target mitochondria and induce cell apoptosis such as betulinic acid have been described (Fulda, S. et al., J. Biol. Chem. 18; 273 (51): 33942-8, 1998; Pezzuto et al. U.S. Patent Application Publication No. 20030186945). Costantini et al. reviews the mechanism of inducing apoptosis through mitochondrial destruction by alteration of mitochondrial membrane permeability and/or changes in the permeability transition pore complex (PTPC) and lists cytotoxic agents that target mitochondria to induce apoptosis (J. Natl. Cancer Inst. 92(13):1042-53, 2000).
The use of a single HAFP/anticancer agent conjugate (i.e. HAFP-estrone-doxorubicin conjugate) is considered to be a limiting factor in the treatment of malignant neoplasms due to the fact that many different types of cancer are refractory to chemotherapy and are said to exhibit multi-drug resistance (MDR) (Lehnert M., Eur. J. Cancer, 32A:912-920, 1996; Germann U. A., Eur. J. Cancer, 32A:927-944, 1996). Moreover, a number of anticancer agents are alkylating agents and antibiotics which induce tumour cell death by targeting DNA and thus, largely rely on an intact p53 signaling pathway (Bykov, V. J. et al., Nat. Med. 8(3):282-8, 2002). Given the large number of tumours that lack functional p53, these treatments are often ineffective.
There is therefore a need for improved mechanisms of delivering cytotoxic agents to cancer cells that are easily derived, inexpensive to produce, deliverable by non-invasive means and both efficient and specific in killing cancer cells.
The present invention may provide one or more of the foregoing advantages or other advantages which will become apparent to persons skilled in the art after review of the present application.